Food

Chemistry
Food Chemistry 98 (2006) 545–550
www.elsevier.com/locate/foodchem

Antioxidant properties of dried product of Ôhaba-noriÕ, an

edible brown alga, Petalonia binghamiae (J. Agaradh) Vinogradova
Takashi Kuda *, Tomoko Hishi, Sayuri Maekawa
Department of Food Science, Ishikawa Prefectural University, Suematsu 1-308, Nonoichi, Ishikawa 921-8836, Japan

Received 14 February 2005; received in revised form 15 June 2005; accepted 15 June 2005

Abstract

Dried Ôhaba-noriÕ Petalonia binghamiae, a brown alga, is a traditional food in the fisheries towns in Japan. To determine the
antioxidant properties of the dried P. binghamiae, assays for antioxidant activities, including ferrous-reducing power, ferrous ion
chelating, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging and scavenging of a superoxide anion radical-generated by
non-enzymatic system were tested in this study. A water extract solution contained total phenols at about 75 lmol phloroglucinol
equivalents/g dry sample and showed strong antioxidant activities in the reducing power, DPPH radical and superoxide anion
radical scavenging assays. The antioxidant activities were detected in high-molecular (>100 kDa), 10–30 kDa, and low-molecular
(<5 kDa) fractions and were correlated with, not only phenolic compounds, but also brown compounds. The radical- scavenging
activities were increased by heat treatment at 121 C for 1 h. These results suggest that P. binghamiae is both a useful seafood
and a healthy food with antioxidant activity.
 2005 Elsevier Ltd. All rights reserved.

Keywords: Petalonia binghamiae; Antioxidant property; Radical scavenging activity; Reducing power

1. Introduction Oxidative modification of DNA, proteins, lipid and

A brown alga, Petalonia binghamiae (J. Agaradh)
Vinogradova called Ôhaba-noriÕ in Japan, is widely dis-
tributed in the Pacific, for example along the coasts of
Japan, China and West of the USA (Segawa, 1996).
The shape of P. binghamiae is an aggregate of several
leaves that are 15–50 mm in width and 100–250 mm in
length. Although P. binghamiae grows well along many
coasts of Japan and other countries, it is consumed as an
edible alga and traditional food only in the fisheries
town areas. Usually, the alga is eaten after drying and
roasting lightly, like a dried product of ÔnoriÕ Porphyra
spp., a red alga, that is one of the major algal products
(Kitamura, Myouga, & Kamei, 2002).
*
Corresponding author. Tel.: +81 76 227 7220; fax: +81 76 227
7410.
E-mail address: kuda@ishikawa-c.ac.jp (T. Kuda).
small cellular molecules by reactive oxygen species
(ROS) plays a role in a wide range of common diseases
and age-related degenerative conditions (Borek, 1993).
These include cardiovascular disease, inflammatory con-
ditions, and neurodegenerative diseases, such as Alzhei-
merÕs disease (Richardson, 1993), mutations and cancer
(Byress & Guerrero, 1995). Though there are publica-
tions about the antioxidant activity of seaweeds (Yan,
Nagata, & Fan, 1998), there are few reports about anti-
oxidant activities in dried algal products (Kuda,
Tsunekawa, Hishi, & Araki, 2005), and these studies
are mainly confined to non-edible and/or fresh raw sea-
weeds. It is reported that drying and storage decrease
the antioxidant compounds and activities (Araki, 1983).
There are some reports about fucoxanthin-related
compounds (Mori et al., 2004), including retinoic acid
(vitamin A) in P. binghamiae. These compounds have
inhibitory activities against mammalian replicative

0308-8146/$ - see front matter  2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2005.06.023
546 T. Kuda et al. / Food Chemistry 98 (2006) 545–550
DNA polymerases (Murakami et al., 2002). However,
there are hardly any reports about any functional activ-
ity, such as antioxidant activity, in dried products of P.
binghamiae.
The aim of the present work was to evaluate the prof-
itable properties of P. binghamiae for human food. We
investigated the antioxidant activities of water extract
and ethanol extract by ferrous-reducing power assay,
ferrous ion chelating assay, DPPH radical assay, and
superoxide anion-scavenging assay. These antioxidant
assays employ methodology widely used for plants and
processed foods. Effects of heating on the antioxidant
activities were also examined.
2. Materials and methods
2.1. Material
P. binghamiae was harvested in Wajima city (located
in the temperate zone and facing the Sea of Japan),
Ishikawa, Japan in April, 2004. The harvested material
was spread in a mesh bottom frame about 25 · 25 cm
and dried like ÕnoriÕ. The dried product was purchased
from a retail shop in Wajima and used in this study.
2.2. Chemicals
(+)-Catechin (CA), Folin–CiocalteuÕs phenol reagent,
the stable radical DPPH, nitroblue tetrazolium salt
(NBT), phenazine methosulphate (PMS), 3-(2-pyridyl)-
5,6-di(p-sulfophenyl)-1,2,4-triazine, disodium salt (ferro-
zine) and ethylendiaminetetraacetic acid (EDTA) were
purchased from Sigma–Aldrich Co. (St. Louis, MO).
Phloroglucinol dihydrate (PG) and Potassium ferricya-
nide were purchased from Wako Chemicals Co., Ltd.
(Osaka, Japan). Other reagents were of analytical grade.
2.3. Preparation of sample extract
The dried product sample (2 g) was weighed and
50 ml of distilled water or ethanol were added. The
water extract (WE) and ethanol extract (EE) solutions
were collected after shaking for 1 h at room temperature
and centrifugation (2220 · 10 min). The sample prepara-
tion was replicated three times. The WE and EE solu-
tions were dark brown and dark green, respectively.
A portion (6 ml) of the WE solution was fractionated
into six fractions (each 6 ml) of molecular weights, that
were >100, 50–100, 30–50, 10–30, 5–10 kDa, and
<5 kDa, using an ultrafiltration system, VIVASPIN 6
(Sartorius AG, Goettingen, Germany).
To determine the effect of heat treatment on the anti-
oxidant activities, the WE and EE were extracted at
85 C and 121 C (WE85, WE121, EE85 and EE121,
respectively).
2.4. Determination of the amount of total phenolic
compounds, brown compounds, protein, saccharides and
minerals
Total phenolic compound concentrations were deter-
mined as described previously (Kuda et al., 2005; Oki
et al., 2002). Briefly, 0.4 ml of 10% Folin–Chiocalteu
solution was added to 0.2 ml of a sample solution. After
an interval of 3 min, 0.8 ml of a 10% sodium carbonate
was added. The mixture was allowed to stand for 30 min
at ambient temperature and the absorbance was then
measured at 750 nm. The phenolic content was ex-
pressed as PG equivalent (Eq). To determine the relative
content of brown compounds in P. binghamiae, 0.2 ml of
WE was put into a microplate well and then absorbance
was measured at 490 nm with reference to absorbance at
655 nm.
Protein content in the individual fractions was mea-
sured with a DC protein assay kit (Bio-Rad, CA). Total
saccharide was determined by the phenol–sulfuric meth-
od (Dubois, Gilles, Hamilton, Rebens, & Smith, 1956).
Salinity was measured by a salt meter (ES-421, Atago,
Tokyo). Concentrations of sodium and potassium were
determined by ion meters (C-122 and C-131, Horiba,
Kyoto). Calcium and magnesium were measured with
commercially available kits (for Ca and Mg Test-Wako
series, Wako Pure Chemical, Osaka).
2.5. Reducing power
Total reducing power was determined as described by
Zhu, Hackman, Ensunsa, Holt, and Keen (2002), but
modified slightly. Briefly, each 0.2 ml of the sample solu-
tion was mixed with 0.2 ml of phosphate buffer (0.2 M,
pH 7.2) and 0.2 ml of 1% potassium ferricyanide. The
mixture was then incubated at 50 C for 20 min. After-
wards, 0.2 ml of 10% trichloroacetic acid was added to
mixture. Finally, 0.125 ml of the mixture and 0.125 ml
distilled water were put into a 96-well microplate and
0.02 ml of 0.1% FeCl3 was added. Increased absorbance
at 655 nm of the reaction mixture indicated increased
reducing power. PG was used as positive control.
2.6. Ferrous ion chelating activity
The method of Decker and Welch (1990) was used. To
a sample solution (0.1 ml), distilled water (0.1 ml) and
0.5 mM FeCl2 (0.025 ml) were added. After measurement
of absorbance at 550 nm, 2.5 mM ferrozine was added.
After 20 min at room temperature, the absorbance was
measured. EDTA was used as positive control.
2.7. DPPH radical-scavenging activity
DPPH radical-scavenging activity was determined by
the method of Blois (1958) with slight modification.
 T. Kuda et al. / Food Chemistry 98 (2006) 545–550
Thirty minutes after adding 1 mM DPPH/methanol
solution (0.025 ml) to the sample solutions (0.2 ml),
absorbance was measured at 550 nm. If the mixture
was turbid, the absorbance was measured after
centrifugation.
2.8. Superoxide anion radical-scavenging activity
The non-enzymatic generation of superoxide anion
was measured by the method of Robak and Gryglewski
(1988). Sample solution (0.1 ml) was treated with 0.1 ml
of 0.1 M phosphate buffer (pH 7.2), 0.025 ml of 2 mM
NADH and 0.025 ml of 0.5 mM NBT, and absorbance
at 550 nm was measured as a blank value. After a
3 min incubation with 0.025 ml of 0.03 mM PMS, the
absorbance was again measured.
3. Results and discussion
3.1. Chemical compounds in extract solutions
The total phenolic contents in WE and EE were 73.5
and 21.8 lmol PG Eq/g dry sample, respectively (Table
1). The other main compounds in WE were saccharides
(polysaccharides). Protein and mineral contents in WE
were not so high. Potassium content was four times
higher than sodium content.
3.2. Antioxidant properties of the water and ethanol
extract solutions
Ferrous-reducing power, DPPH radical scavenging
activity and superoxide anion radical scavenging activity
of WE were higher than that of EE (Table 2). The phe-
nolic content, ferrous-reducing power and radical scav-
enging activity in WE was higher than that of
common brown algae, such as Fucus, Laminaria, Unda-
ria, (Jiménez-Escrig, Jiménez-Jiménez, Pulido, & Saura-
Calixto (2001)), besides of ÔkajimeÕ Ecklonia cava (Nagai
& Yukimoto (2003)).
Table 1
Chemical compounds in extractions of dried Ôhaba-noriÕ P. binghamiae
WE
Total phenolic compounds 75.3 ± 7.5
(lmol PG Eq/g dry sample)
Soluble saccharide (mg/g dry sample) 78.1 ± 15.2
Soluble protein (mg/g dry sample) 5.15 ± 0.37
Salinity (mg/g dry sample) 53 ± 1
Na 3.3 ± 0.1
K 11.3 ± 0.3
Mg 1.68 ± 0.05
Ca 1.52 ± 0.19
WE, water extract solution; EE, ethanol extract solution.
PG, phloroglucinol; Eq, equivalent.
Values are means and SD (n = 3).
547
Table 2
Antioxidant properties of dried Ôhaba-noriÕ P. binghamiae
WE EE
Ferrous-reducing power 94.2 ± 2.9 18.9 ± 0.1
(lmol PG Eq/g dry sample)
Ferrous ion chelating activity 2.32 ± 0.33 <2
(lmol EDTA Eq/g dry sample)
DPPH radical-scavenging 2.73 ± 0.49 0.53 ± 0.16
(mmol PG Eq/g dry sample)
Superoxide anion scavenging 80.0 ± 1.9 5.9 ± 1.7
(lmol CA Eq/g dry sample)
WE, water extract solution; EE, ethanol extract solution.
PG, phloroglucinol; Eq, equivalent; CA, (+) catechin.
Values are means and SD (n = 3).
DPPH has been used extensively as a radical to eval-
uate reducing substances (Cotelle et al. (1996)). In most
cases, irrespective of the stage in the oxidative chain in
which the antioxidant action is assessed, most non-enzy-
matic antioxidative activity, such as scavenging free rad-
icals or inhibition of peroxidation, is mediated by redox
reactions (Zhu et al. (2002)). In most organisms, super-
oxide anion radical is converted to hydrogen peroxide
by superoxide dismutase. In the absence of transition
metal ions, hydrogen peroxide is fairly stable. However,
hydroxyl radicals can be formed by the reaction of
superoxide with hydrogen peroxide in the presence of
metal ions, usually ferrous or copper (Macdonald, Gal-
ley, & Webster (2003)).
Ferrous ion chelating activity of both WE and EE
was not so high comparing with Ôkayamo-noriÕ Scytosi-
phon lomentaria (Kuda et al. (2005)). The metal binding
capacities of dietary fibers are well known and the inhib-
itory effects on ferrous absorption of algal dietary fibers
have been reported (Harmuth-Hoene & Schelenz
(1980)). It is considered that the amount and/or ferrous
binding capacities of water-soluble polysaccharides,
such as alginate, fucoidan and laminaran, in P. bingh-
amiae were lower than ones of S. lomentaria.
3.3. Antioxidant properties of individual P. binghamiae
molecular weight fractions
As summarized in Table 1, WE of P. binghamiae was
rich in phenolic compounds. With respect to the six
EE molecular weight fractions isolated, the greatest amount
of phenolic compounds was found in the >5 kDa, and
21.8 ± 0.3
>100 kDa fractions (Fig. 1(a)). The lowest amount of
– phenolic compounds was measured in the 50–100- and
– 30–50-kDa fractions.
– Absorbance at 490 nm referenced by the absorbance at
–
655 nm was used as brown compounds (Fig. 1(b)). About
–
– 60%, 20% and 10% of the brown compounds were in the
– >100-, 10–30- and <5-kDa fraction, respectively.
As shown in Fig. 1(c), the highest ferrous-reducing
power was observed in the >100 kDa, followed by <5,
10–30 and 5–10-kDa fractions. This result is thought
548 T. Kuda et al. / Food Chemistry 98 (2006) 545–550

Fig. 1. Antioxidant properties of individual molecular weight fractions from dried P. binghamiae. (a) Amount of total phenolic compounds. (b)
Relative content of brown compounds. (c) Ferrous-reducing power. (d) Ferrous ion chelating activity. (e) DPPH radical scavenging activity. (f)
Scavenging activity in non-enzymatic assay. Sample solution volumes in the assays c–f were 0.1, 0.1, 0.05 and 0.05 ml, respectively. Values are means
and SD (n = 3).

to correlate with the content of brown compounds
rather than with the content of phenolic compounds,
though many researchers have suggested that there
may be a relationship between the amount of total phe-
nolic compound and reducing power (Jiménez-Escrig
et al., 2001; Zue et al., 2002).
The highest ferrous ion chelating activity was ob-
served in the 10–30-kDa fraction, followed by <5-kDa
fraction (Fig. 1(d)). Interestingly, the chelating activity
of the 10–30-kDa fraction (95%) was higher than that
of the WE (52%) at the same sample solution volume
(0.1 ml) in this assay.
The highest DPPH scavenging activity was shown in
the <5-kDa fraction, followed by <100- and 10–30-kDa
fractions (Fig. 1(e)). This result is thought to correlate
with the content of phenolic compounds rather than
with the content of brown compounds. As with reducing
power, it has been reported that the amount of DPPH-
scavenging activity is dependent on the phenolic concen-
tration of the algal samples (Jiménez-Escrig et al., 2001).
The highest superoxide anion radical scavenging
activity was shown in the >100-kDa fraction followed
by the 10–30- and <5-kDa fractions (Fig. 1(f)). This re-
sult is similar to that of the ferrous-reducing power
(Fig. 1(c)).
3.4. Effect of extraction temperature on the antioxidant
properties
To determine the effect of heating on the antioxida-
tive activity of P. binghamiae, WE and EE were obtained
from dried P. binghamiae at room temperature, 85 and
121 C for 1 h.
The total phenolic compounds in WE extracted at
121 C (WE121) was 65% higher than that of WE ex-
tracted at room temperature (WERT) (Fig. 2(a)). On
the other hand, the content in WE heated at 85 C
(WE85) was decreased slightly. The phenolic content
in EE was not so affected by the extraction tempera-
ture. The brown compound was increased about 2.5
 T. Kuda et al. / Food Chemistry 98 (2006) 545–550 549

Fig. 2. Effect of heating on the antioxidant activities of water extract (open square) and ethanol extract (semi-closed square) solutions from dried P.
binghamiae. (a) Amount of total phenolic compounds. (b) Relative content of brown compounds. (c) Ferrous-reducing power. (d) Ferrous ion
chelating activity. (e) DPPH radical scavenging activity. (f) Scavenging activity in non-enzymatic assay. Sample solution volumes in the assays c–f
were 25, 50, 1.25 and 12.5 ll, respectively. R.T., room temperature. Values are means and SD (n = 3).

times in WE121 (Fig. 2(b)). It is considered that the
brown compound was generated by the Maillard
reaction.
As shown in Fig. 2(c), the reducing power was not in-
creased in WE121. Furthermore, the reducing power de-
creased by 35% in WE85. This result may be related to
phenolic compound content. Jiménez-Escrig et al.
(2001) reported that the phenolic content and reducing
power in Fucus were decreased by drying at 50 C for
48 h, and storage at room temperature.
The chelating activities were about 65% and 100% in-
creased in WE85 and WE121, respectively (Fig. 2(d)).
Perhaps, the increased activity was brought about by
increasing solubility of polysaccharides. We had re-
ported that the crude alginate and fucoidan from Scyto-
siphon lomentaria, heated at 121 C for 15 min, have the
ferrous ion binding activity (Kuda et al., 2005).
DPPH radical scavenging activities in WE were 20%
and 50% increased by heating at 85 and 121 C, respec-
tively (Fig. 2(e)). On the other hand, the activity in EE
was decreased slightly by the heat treatment. Superoxide
anion radical-scavenging activity in WE was also clearly
increased by the heating (Fig. 2(f)).
It is reported that drying and storage decrease antiox-
idant compounds, such as ascorbic acid and fucoxan-
tins, and their activities (Araki, 1983). However, there
are many reports about radical-scavenging activity of
brown compounds (pigments) induced by Maillard reac-
tion (Jing & Kitts, 2002; Morales & Babbel, 2002). It is
considered that the brown compounds having radical
scavenging activity are generated by the Maillard reac-
tion in WE during the heating.
Although there are several reports about antioxidant
activity of raw edible brown algae, such as ÔkombuÕ
550 T. Kuda et al. / Food Chemistry 98 (2006) 545–550
Laminaria, ÔwakameÕ Undaria and ÔkajimeÕ Ecklonia
(Jiménez-Escrig et al., 2001; Kang et al., 2004; Yan
et al., 1998), we believe that the algal foods circulating
as dried product should be examined after the drying pro-
cess. Usually, these brown algae are dried and eaten after
swelling with 20–40 and more volume of water. On the
other hand, P. binghamiae is eaten after drying and only
light roasting.
In summary, our observations demonstrate that WE
and its >100- <5- and also 10–30-kDa fractions have
reducing power and radical scavenging activity. Further-
more, the radical scavenging and ferrous-chelating activ-
ities are promoted by heat treatment such as retortring.
In general, it is considered that the antioxidant activities
depend on the phenolic and/or brown compounds.
However, the antioxidant activities of sulfated polysac-
charides, such as fucoidan have been reported (Kuda
et al., 2005; Ruperez, Ahrazem, & Leal, 2002). From
the results of the study and the processes, we consider
that P. binghamiae is a useful healthy sea vegetable hav-
ing antioxidant activities. It is necessary to begin exper-
iments and purification of antioxidants in WE. We are
especially interested in the 10–30 kDa fraction, of P.
binghamiae. These studies are now in progress.
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